Bart van Oort

Molecular basis of photoprotection and control of photosynthetic light-harvesting

In order to maximize their use of light energy in photosynthesis, plants have molecules that act as light-harvesting antennae, which collect light quanta and deliver them to the reaction centres, where energy conversion into a chemical form takes place. The functioning of the antenna responds to the extreme changes in the intensity of sunlight encountered in nature. In shade, light is efficiently harvested in photosynthesis. However, in full sunlight, much of the energy absorbed is not needed and there are vitally important switches to specific antenna states, which safely dissipate the excess energy as heat. This is essential for plant survival, because it provides protection against the potential photo-damage of the photosynthetic membrane. But whereas the features that establish high photosynthetic efficiency have been highlighted, almost nothing is known about the molecular nature of the dissipative states. Recently, the atomic structure of the major plant light-harvesting antenna protein, LHCII, has been determined by X-ray crystallography. Here we demonstrate that this is the structure of a dissipative state of LHCII. We present a spectroscopic analysis of this crystal form, and identify the specific changes in configuration of its pigment population that give LHCII the intrinsic capability to regulate energy flow. This provides a molecular basis for understanding the control of photosynthetic light-harvesting.

Aggregation of Light-Harvesting Complex II leads to formation of efficient excitation energy traps in monomeric and trimeric complexes

Non‐photochemical quenching (NPQ) protects plants against photodamage by converting excess excitation energy into harmless heat. In vitro aggregation of the major light‐harvesting complex (LHCII) induces similar quenching, the molecular mechanism of which is frequently considered to be the same. However, a very basic question regarding the aggregation‐induced quenching has not been answered yet. Are excitation traps created upon aggregation, or do existing traps start quenching excitations more efficiently in aggregated LHCII where trimers are energetically coupled? Time‐resolved fluorescence experiments presented here demonstrate that aggregation creates traps in a significant number of LHCII trimers, which subsequently also quench excitations in connected LHCIIs.

Effect of Antenna-Depletion in Photosystem II on Excitation Energy Transfer in Arabidopsis thaliana

The role of individual photosynthetic antenna complexes of Photosystem II (PSII) both in membrane organization and excitation energy transfer have been investigated. Thylakoid membranes from wild-type Arabidopsis thaliana, and three mutants lacking light-harvesting complexes CP24, CP26, or CP29, respectively, were studied by picosecond-fluorescence spectroscopy. By using different excitation/detection wavelength combinations it was possible for the first time, to our knowledge, to separate PSI and PSII fluorescence kinetics. The sub-100 ps component, previously ascribed entirely to PSI, turns out to be due partly to PSII. Moreover, the migration time of excitations from antenna to PSII reaction center (RC) was determined for the first time, to our knowledge, for thylakoid membranes. It is four times longer than for PSII-only membranes, due to additional antenna complexes, which are less well connected to the RC. The results in the absence of CP26 are very similar to those of wild-type, demonstrating that the PSII organization is not disturbed. However, the kinetics in the absence of CP29 and, especially, of CP24 show that a large fraction of the light-harvesting complexes becomes badly connected to the RCs. Interestingly, the excited-state lifetimes of the disconnected light-harvesting complexes seem to be substantially quenched.

Picosecond Fluorescence Of Intact And Dissolved PSI-LHCI Crystals

Over the past several years, many crystal structures of photosynthetic pigment-protein complexes have been determined, and these have been used extensively to model spectroscopic results obtained on the same proteins in solution. However, the crystal structure is not necessarily identical to the structure of the protein in solution. Here, we studied picosecond fluorescence of photosystem I light-harvesting complex I (PSI-LHCI), a multisubunit pigment-protein complex that catalyzes the first steps of photosynthesis. The ultrafast fluorescence of PSI-LHCI crystals is identical to that of dissolved crystals, but differs considerably from most kinetics presented in the literature. In contrast to most studies, the data presented here can be modeled quantitatively with only two compartments: PSI core and LHCI. This yields the rate of charge separation from an equilibrated core (22.5 +/- 2.5 ps) and rates of excitation energy transfer from LHCI to core (k(LC)) and vice versa (k(CL)). The ratio between these rates, R = k(CL)/k(LC), appears to be wavelength-dependent and scales with the ratio of the absorption spectra of LHCI and core, indicating the validity of a detailed balance relation between both compartments. k(LC) depends slightly but nonsystematically on detection wavelength, averaging (9.4 +/- 4.9 ps)(-1). R ranges from 0.5 (<690 nm) to approximately 1.3 above 720 nm.

(Sub)-Picosecond Spectral Evolution of Fluorescence Studied with a Synchroscan Streak-Camera System and Target Analysis

Summary A synchroscan streak camera in combination with a spectrograph can simultaneously record temporal dynamics and wavelength of fl uorescence representable as an image with time and wavelength along the axes. The instrument response width is about 1% of the time range (of typically 200 ps to 2 ns). The spectral window of 250 nm may lie between 250 and 850 nm. Such spectrotemporal measurements using low excitation intensities

Picosecond Fluorescence Relaxation Spectroscopy of the Calcium-Discharged Photoproteins Aequorin and Obelin

Addition of calcium ions to the Ca(2+)-regulated photoproteins, such as aequorin and obelin, produces a blue bioluminescence originating from a fluorescence transition of the protein-bound product, coelenteramide. The kinetics of several transient fluorescent species of the bound coelenteramide is resolved after picosecond-laser excitation and streak camera detection. The initially formed spectral distributions at picosecond-times are broad, evidently comprised of two contributions, one at higher energy (approximately 25,000 cm(-1)) assigned as from the Ca(2+)-discharged photoprotein-bound coelenteramide in its neutral state. This component decays much more rapidly (t(1/2) approximately 2 ps) in the case of the Ca(2+)-discharged obelin than aequorin (t(1/2) approximately 30 ps). The second component at lower energy shows several intermediates in the 150-500 ps times, with a final species having spectral maxima 19 400 cm(-1), bound to Ca(2+)-discharged obelin, and 21 300 cm(-1), bound to Ca(2+)-discharged aequorin, and both have a fluorescence decay lifetime of 4 ns. It is proposed that the rapid kinetics of these fluorescence transients on the picosecond time scale, correspond to times for relaxation of the protein structural environment of the binding cavity.

PSI–LHCI of Chlamydomonas reinhardtii: Increasing the absorption cross section without losing efficiency

Photosystem I (PSI) is an essential component of photosynthetic membranes. Despite the high sequence and structural homologies, its absorption properties differ substantially in algae, plants and cyanobacteria. In particular it is characterized by the presence of low-energy chlorophylls (red forms), the number and the energy of which vary in different organisms. The PSI–LHCI (PSI–light harvesting complex I) complex of the green alga Chlamydomonas reinhardtii (C.r.) is significantly larger than that of plants, containing five additional light-harvesting complexes (together binding ≈ 65 chlorophylls), and contains red forms with higher energy than plants. To understand how these differences influence excitation energy transfer and trapping in the system, we studied two PSI–LHCI C.r. particles, differing in antenna size and red-form content, using time-resolved fluorescence and compared them to plant PSI–LHCI. The excited state kinetics in C.r. shows the same average lifetime (50 ps) as in plants suggesting that the effect of antenna enlargement is compensated by higher energy red forms. The system equilibrates very fast, indicating that all Lhcas are well-connected, despite their long distance to the core. The differences between C.r. PSI–LHCI with and without Lhca2 and Lhca9 show that these Lhcas bind red forms, although not the red-most. The red-most forms are in (or functionally close to) other Lhcas and slow down the trapping, but hardly affect the quantum efficiency, which remains as high as 97% even in a complex that contains 235 chlorophylls.

A Hidden State in Light-Harvesting Complex II Revealed By Multipulse Spectroscopy

Light-harvesting complex II (LHCII) is pivotal both for collecting solar radiation for photosynthesis, and for protection against photodamage under high light intensities (via a process called nonphotochemical quenching, NPQ). Aggregation of LHCII is associated with fluorescence quenching, and is used as an in vitro model system of NPQ. However, there is no agreement on the nature of the quencher and on the validity of aggregation as a model system. Here, we use ultrafast multipulse spectroscopy to populate a quenched state in unquenched (unaggregated) LHCII. The state shows characteristic features of lutein and chlorophyll, suggesting that it is an excitonically coupled state between these two compounds. This state decays in approximately 10 ps, making it a strong competitor for photodamage and photochemical quenching. It is observed in trimeric and monomeric LHCII, upon re-excitation with pulses of different wavelengths and duration. We propose that this state is always present, but is scarcely populated under low light intensities. Under high light intensities it may become more accessible, e.g. by conformational changes, and then form a quenching channel. The same state may be the cause of fluorescence blinking observed in single-molecule spectroscopy of LHCII trimers, where a small subpopulation is in an energetically higher state where the pathway to the quencher opens up.

The High Efficiency of Photosystem I in the Green Alga Chlamydomonas reinhardtii Is Maintained after the Antenna Size Is Substantially Increased by the Association of Light-harvesting Complexes II.

Background: Photosystems acclimate to different light conditions modulating their antenna size. Results: In PSI-LHCI-LHCII, energy transfer from LHCII to Photosystem I is relatively slow but still very efficient. Conclusion: The association of LHCII to PSI increases its absorption cross section by ∼50%, maintaining ∼96% quantum efficiency. Significance: The antenna size of PSI can be increased considerably without sacrificing its efficiency. Photosystems (PS) I and II activities depend on their light-harvesting capacity and trapping efficiency, which vary in different environmental conditions. For optimal functioning, these activities need to be balanced. This is achieved by redistribution of excitation energy between the two photosystems via the association and disassociation of light-harvesting complexes (LHC) II, in a process known as state transitions. Here we study the effect of LHCII binding to PSI on its absorption properties and trapping efficiency by comparing time-resolved fluorescence kinetics of PSI-LHCI and PSI-LHCI-LHCII complexes of Chlamydomonas reinhardtii. PSI-LHCI-LHCII of C. reinhardtii is the largest PSI supercomplex isolated so far and contains seven Lhcbs, in addition to the PSI core and the nine Lhcas that compose PSI-LHCI, together binding ∼320 chlorophylls. The average decay time for PSI-LHCI-LHCII is ∼65 ps upon 400 nm excitation (15 ps slower than PSI-LHCI) and ∼78 ps upon 475 nm excitation (27 ps slower). The transfer of excitation energy from LHCII to PSI-LHCI occurs in ∼60 ps. This relatively slow transfer, as compared with that from LHCI to the PSI core, suggests loose connectivity between LHCII and PSI-LHCI. Despite the relatively slow transfer, the overall decay time of PSI-LHCI-LHCII remains fast enough to assure a 96% trapping efficiency, which is only 1.4% lower than that of PSI-LHCI, concomitant with an increase of the absorption cross section of 47%. This indicates that, at variance with PSII, the design of PSI allows for a large increase of its light-harvesting capacities.

Profiling of dynamics in protein–lipid–water systems: a time-resolved fluorescence study of a model membrane protein with the label BADAN at specific membrane depths

Profiles of lipid-water bilayer dynamics were determined from picosecond time-resolved fluorescence spectra of membrane-embedded BADAN-labeled M13 coat protein. For this purpose, the protein was labeled at seven key positions. This places the label at well-defined locations from the water phase to the center of the hydrophobic acyl chain region of a phospholipid model membrane, providing us with a nanoscale ruler to map membranes. Analysis of the time-resolved fluorescence spectroscopic data provides the characteristic time constant for the twisting motion of the BADAN label, which is sensitive to the local flexibility of the protein–lipid environment. In addition, we obtain information about the mobility of water molecules at the membrane–water interface. The results provide an unprecedented nanoscale profiling of the dynamics and distribution of water in membrane systems. This information gives clear evidence that the actual barrier of membranes for ions and aqueous solvents is located at the region of carbonyl groups of the acyl chains.